Pseudomonas syringae pv. syringae által termelt lipopeptidek és a sejtmembrán közötti kölcsönhatások vizsgálata = Investigation of lipopeptides and cell membrane interactions

Az OTKA projekt keretében három, különböző szerkezetű és biológiai aktivitású, antifungális ciklusos lipodepsipeptid, a syringomycin-E (SR-E), a syringotoxin (ST-B) és a syringopeptin-25A (SP-25A) vegyület, hatásmechanizmusát vizsgáltuk atomi szintű molekula dinamikai (MD) szimulációval. A projekt első két évében a három CLP molekula hidrofil és hidrofób közegben kialakuló háromdimenziós (3D) szerkezetét határoztuk meg. Eredményeink összhangban vannak az irodalomból ismert kísérleti megállapításokkal, amelyeket két közleményben foglaltunk össze. Az MD számolások eredményeként meghatározott és jellemzett konformereket építettük be a sejt membránt modellező, különböző típusú és összetételű lipid kettősrétegekbe, amelyeket a projekt harmadik évében építettünk fel. A kettősrétegek megszerkesztése önmagában nem leközölhető eredmény, azonban várhatóan egy lipid adatbázisba bekerülve mások által is alkalmazhatóvá válhatnak. A projekt negyedik évében az összeépített, különböző összetételű és méretű, peptid-lipid kettősréteg rendszereken további MD szimulációt végeztünk, hogy a peptid-lipid kölcsönhatás, továbbá a peptid és a lipid szerkezetében bekövetkező változásokat, a pórus szerkezetét és kialakulását molekuláris szinten értelmezhessük. Megállapítottuk a peptidek szerkezetében és a kettősréteg rendezettségében bekövetkező változásokat, továbbá hogy a pórus kialakulásában a peptidek mellett a lipid molekulák is jelentős szerepet játszanak. | Syringomycin-E (SR-E), the syringotoxin (ST-B) and the syringopeptin-25A (SP-25A) are antifungal cyclic lipodepsipeptides (CLPs) produced by certain strains of the bacterium Pseudomonas syringae pv. syringae. Their primary biological target is the plasma membrane, where their form channels. In this project, we investigated in atomic detail the molecular feature of CLPs in water, octane and different composition of lipid bilayers by atomistic molecular dynamics simulations. The 3D structures of CLPs obtained after long (200ns) MD simulation in hydrophobic and hydrophilic environment are in agreement with published experimental data and the results are summarized in two papers. These structures are built into different lipid bilayers are used to investigate the interactions of CLPs with lipid bilayers, the mechanism of pore formation and the structure of pore formed by CLPs

Water chemistry analysis in the sediment of Baradla Cave, Hungary

Abstract Water was observed in the sediment of Baradla Cave, located in Northeast Hungary. In order to investigate its characteristics wells were drilled. Hydrochemical samples were taken directly from the wells and from the cave stream on several occasions between November 2009 and April 2010. In February 2010 there was an opportunity to observe how the chemical composition of the waters of the creeks and the sediments altered during the snow melt. Several chemical parameters of the samples were analyzed. Based on the results of the hydrochemical analyses cluster analysis was applied to define the relationship between the sampling points. Discriminant analysis was conducted to verify the classification. As a result of the classification, the water of the observation wells in the sediment proved to be distinct from the water of the cave's creek and the springs on the surface. Research shows that there is no permanent connection between the water in the cave sediment and the water of the cave creek in the cave water system

Low-mutation-rate, reduced-genome Escherichia coli: an improved host for faithful maintenance of engineered genetic constructs

<p>Abstract</p> <p>Background</p> <p>Molecular mechanisms generating genetic variation provide the basis for evolution and long-term survival of a population in a changing environment. In stable, laboratory conditions, the variation-generating mechanisms are dispensable, as there is limited need for the cell to adapt to adverse conditions. In fact, newly emerging, evolved features might be undesirable when working on highly refined, precise molecular and synthetic biological tasks.</p> <p>Results</p> <p>By constructing low-mutation-rate variants, we reduced the evolutionary capacity of MDS42, a reduced-genome <it>E. coli </it>strain engineered to lack most genes irrelevant for laboratory/industrial applications. Elimination of diversity-generating, error-prone DNA polymerase enzymes involved in induced mutagenesis achieved a significant stabilization of the genome. The resulting strain, while retaining normal growth, showed a significant decrease in overall mutation rates, most notably under various stress conditions. Moreover, the error-prone polymerase-free host allowed relatively stable maintenance of a toxic methyltransferase-expressing clone. In contrast, the parental strain produced mutant clones, unable to produce functional methyltransferase, which quickly overgrew the culture to a high ratio (50% of clones in a 24-h induction period lacked functional methyltransferase activity). The surprisingly large stability-difference observed between the strains was due to the combined effects of high stress-induced mutagenesis in the parental strain, growth inhibition by expression of the toxic protein, and selection/outgrowth of mutants no longer producing an active, toxic enzyme.</p> <p>Conclusions</p> <p>By eliminating stress-inducible error-prone DNA-polymerases, the genome of the mobile genetic element-free <it>E. coli </it>strain MDS42 was further stabilized. The resulting strain represents an improved host in various synthetic and molecular biological applications, allowing more stable production of growth-inhibiting biomolecules.</p

Testing for Consumer Preferences of Smoked Asian Sea Bass (Barramundi) Filet Products in Hungary

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Alterations of membrane protein expression in red blood cells of Alzheimer's disease patients

Preventive measures, prognosis, or selected therapy in multifactorial maladies, including Alzheimer's disease (AD), require the application of a wide range of diagnostic assays. There is a large unmet need for relatively simple, blood-based biomarkers in this regard. We have recently developed a rapid and reliable flow cytometry and antibody-based method for the quantitative measurement of various red blood cell (RBC) membrane proteins from a drop of blood. Here, we document that the RBC expression of certain membrane proteins, especially that of the GLUT1 transporter and the insulin receptor (INSR), is significantly higher in AD patients than in age-matched healthy subjects. The observed differences may reflect long-term metabolic alterations relevant in the development of AD. These findings may pave the way for a diagnostic application of RBC membrane proteins as relatively stable and easily accessible personalized biomarkers in AD. © 2015 The Authors

Analysis of drip water in an urban karst cave beneath the Hungarian capital (Budapest)

Our geological heritage is increasingly threatened by anthropogenic activity. This is especially true of the Pál-völgyi Cave System beneath Budapest. It is among the 150 longest and at the same time most endangered cave systems in the world. The aims of the study were (i) to set up a monitoring system in the cave, (ii) to track the daily changes in the quality and quantity of drip water, and (iii) to determine the exposure of the cave. Monitoring was conducted at two locations in a shallow area next to a fracture zone (site name: TG) and one lying in a tectonically less disturbed, geologically more homogeneous location 20 m deeper (site YC). The data obtained in respect of 13 variables were assessed using descriptive statistics, principal component- and periodicity analyses. At first glance, it was apparent that the eight water quality parameters differed in quantity between the two sites. Furthermore, using principal component analysis it was shown that in the fractured-shallow setting, anthropogenic activity (external urban pollution, e.g. de-icing, decrease of land cover etc.) is the driving process determining water quality. At the tectonically less fractured site (YC) external influences originating above ground may be added to the natural karst-forming processes. The assessment of drip intensity and electric conductivity again highlighted the differences between the sites in terms of their reaction to precipitation. With regard to diurnal periodicity, although pH and Eh indicated a mature periodic behavior at both sites (covering 56−65 % of the total observed time), at site TG electric conductivity displayed diurnal periodicity over only 21 % of the total time, compared to 56 % at YC. All results pointed towards a conclusion that at site YC daily periodicity and water quality are much more connected to natural processes, while at site TG anthropogenic external influences suppress these.Key words: cave drip water, hydrochemistry, karst, Pál-völgyi Cave System, time series analysis, urban pollution. Analiza prenikle vode v urbani kraški jami pod madžarsko prestolnico (Budimpešta)Naša geološka dediščina je vse bolj ogrožena zaradi človekovih dejavnosti. To še posebej velja za jamski sistem Pál-völgyi pod Budimpešto. Sodi med 150 najdaljših in hkrati najbolj ogroženih jamskih sistemov na svetu. Cilji raziskave so bili (i) vzpostaviti sistem spremljanja izbranih parametrov v jami, (ii) spremljati dnevne spremembe kakovosti in količine prenikle vode, in (iii) določiti izpostavljenost jame onesnaženju. Raziskava je bila izvedena na dveh lokacijah v plitvem območju blizu razpoklinske cone (ime vzorčnega mesta: TG) in na tektonsko manj pretrti in geološko bolj homogeni lokaciji 20 m globlje (ime vzorčnega mesta: YC). Pridobljeni podatki 13 spremenljivk so bili obdelani s pomočjo opisne statistike, analize glavnih komponent in periodičostno analizo. Na prvi pogled je bilo očitno, da se je osem parametrov kakovosti vode kvantitativno razlikovalo med obema mestoma. Poleg tega se je s pomočjo analize glavnih komponent pokazalo, da je mesto v plitvem pretrtem območju pod velikim vplivom antropogenih aktivnosti (urbano onesnaženje, npr. sredstva proti zmrzovanju, zmanjšanje pokrovnih plasti, itd.), ki vplivajo na kakovost vode. Na tektonsko manj preoblikovanem mestu (YC) se zunanji vplivis površja mešajo z naravnimi kraškimi procesi.  Ocena intenzivnosti kapljanja in električne prevodnosti je ponovno izpostavila razlike med vzorčnimi mesti v smislu njunega odziva na padavine. V zvezi z dnevno periodičnostjo je vzorčno mesto TG kazalo dnevno periodičnost več kot 21 % celotnega časa, mesto YC 56 %, čeprav sta pH in Eh izkazali zrelo periodično obnašanje (56−65 % celotnega opazovanega časa) na obeh mestih. Vsi rezultati kažejo na sklep, da je na mestu YC dnevna periodičnost in kakovost vode veliko bolj povezana z naravnimi procesi, medtem ko jih na mestu TG antropogeni zunanji vplivi zadušijo.Ključne besede: jamska prenikla voda, hidrokemija, kras, jamski sistem Pál-völgyi, analiza časovne vrste, urbano onesnaževanje.

Birtokkoncentráció, foglalkoztatás, diverzifikáció és multifunkcionalitás

A statisztikai adatok szerint a magyar mezőgazdaság munkaerő-felhasználása folyamatosan csökken. Cikkünkben rávilágítunk arra az ellentmondásra, ami a deklarált foglalkoztatási célok és a versenyképességet szolgáló gépberuházási támoga¬tások növekedése, valamint hatásai között feszül. E támogatások hozzájárulhatnak ahhoz, hogy a gazdák birtoknövelési törekvéseit erősítsék, s fokozzák a mezőgazdaság munkaerő-kibocsátását. Nem igazán halad a folyamatot potenciálisan mérséklő tevékenységdiverzifikáció sem. Három LEADER kistérségben 104 gazdálkodóra kiterjedő felmérésünkkel feltártuk a gazdák és gazdacsaládok gazdaságfejlesztési motívumait, a gazdaság diverzifikációjának helyzetét, a multifunkciós mezőgazdálkodással kapcsolatos terveiket, s az ezeket segítő és akadályozó tényezőket. A mezőgazdasági termelésen túli funkciókon belül a nem árujellegű közszolgáltatásokkal külön is foglalkoztunk. A vizsgált gazdák természetesnek tekinthető biztonságos gazdaságfejlesztési motívuma mellett a család jobb megélhetése játszott meghatározó szerepet. Ezek realizálásához legtöbben a földvásárlást tartották a legfontosabbnak. A foglalkoztatásban a családtagok kerültek előtérbe, a mások számára történő munkahelyteremtést a legtöbben – még a diverzifikált gazdaságok esetében is – az utolsó helyekre tették. A rugalmas foglalkoztatási formák terjesztését kifejezetten állami feladatnak tekintik. Mindezek felhívják a figyelmet a központilag megfogalmazott célok, a területi és ágazati folyamatok, s a gazdák gondolkodása közötti, a foglalkoztatás vonatkozásában megjelenő ellentmondásokra. --------------------------------------------------------------- Statistics reveal that the number of people employed in Hungarian agriculture is constantly decreasing. Our paper highlights the contradiction between the employment goals announced and the proliferation and effect of machinery acquisition subsidies granted to improve competitiveness. These subsidies may reinforce farmers’ attempts to increase their land, and contribute to the laying off of agricultural workforce. Diversification of operations could potentially counter this process, but its progress is hardly discernible. We surveyed 104 farmers in three LEADER micro-regions to find out about the farm development motives of farmers and farmer families, the status of farm diversification, their plans concerning multifunctional agriculture, as well as the relevant positive and negative factors. Within the functions other than agricultural production, we specifically addressed public services other than the provision of goods. Apart from their natural desire to develop their farm in a safe manner, the farmers interviewed were motivated mostly by a desire to improve the financial situation of the family. Most of them thought that purchasing more land would be the most important step towards this goal. With respect to employment, family members were mentioned, while the creation of employment opportunities for other was ranked very low, even by diversified farms. The promotion of flexible forms of employment was specifically seen a governmental responsibility. The findings highlight the contradictions existing between centrally announced goals, regional and industry processes and the attitude of farmers in the field of employment

Some factors responsible for reductions in employment on farms in Hungary

A decline in the number of people working on farms and in the Annual Work Units per unit area was typical of the second half of the first decade of the 21st century in the member states of the European Union. Hungary is one of the countries where this reduction is twice as high as the average. This can be attributed to a number of factors. Of these, the present paper is concerned on the one hand with farm concentration, the low level of farm diversification and pluriactivity and the desire of farmers to expand their farms, and on the other hand with various aspects of the support policy in Hungary aimed at the economic competitiveness and the diversification of the rural economy. The database on which the work was based was taken partly from the digital and printed publications of EUROSTAT and the Hungarian Central Statistical Office (KSH) and partly from surveys of 104 farmers in three microregions of Hungary. Even before the global economic crisis, the factors in question tended to result in a decline in farm employment in Hungary, especially on individual farms. The means and measures embodied in the agricultural and rural policies proved too few and too weak to counterbalance this trend

Crosstalk between the Arabidopsis Glutathione Peroxidase-Like 5 Isoenzyme (AtGPXL5) and Ethylene

Glutathione peroxidases (GPXs) are important antioxidant enzymes in animals. Plants contain GPX-like (GPXL) enzymes, which-in contrast to GPXs-contain cysteine in their active site instead of selenocysteine. Although several studies proved their importance in development and stress responses, their interaction with ethylene (ET) signalling is not known. Our aim was to investigate the involvement of AtGPXL5 in ET biosynthesis and/or signalling using Atgpxl5 mutant and AtGPXL5 cDNA-overexpressing (OX-AtGPXL5) lines. Four-day-old dark-grown Atgpxl5 seedlings had shorter hypocotyls and primary roots, while OX-AtGPXL5 seedlings exhibited a similar phenotype as wild type under normal conditions. Six-week-old OX-AtGPXL5 plants contained less H2O2 and malondialdehyde, but higher polyamine and similar ascorbate- and glutathione contents and redox potential (E-GSH) than the Col-0. One-day treatment with the ET-precursor 1-aminocyclopropane-1-carboxylic acid (ACC) induced the activity of glutathione- and thioredoxin peroxidases and some other ROS-processing enzymes. In the Atgpxl5 mutants, the E-GSH became more oxidised; parallelly, it produced more ethylene after the ACC treatment than other genotypes. Although the enhanced ET evolution measured in the Atgpxl5 mutant can be the result of the increased ROS level, the altered expression pattern of ET-related genes both in the Atgpxl5 and OX-AtGPXL5 plants suggests the interplay between AtGPXL5 and ethylene signalling

Razvoj i usporedba alternativnih metoda pročišćavanja adalimumaba izravno iz suspenzije stanične kulture

Research background. Protein A affinity chromatography is a well-established method currently used in the pharmaceutical industry. However, the high costs usually associated with chromatographic separation of protein A and the difficulties in continuous operation make the investigation of alternative purification methods very important. Experimental approach. In this study, extraction/back-extraction and precipitation/dissolution methods were developed and optimised. They were compared with protein A and cation exchange chromatography separations in terms of yield of monoclonal antibody (mAb) and amount of residual impurities, such as DNA and host cell proteins, and amount of mAb aggregates. For a comprehensive comparison of the different methods, experiments were carried out with the same cell-free fermentation broth containing adalimumab. Results and conclusions. Protein A and cation exchange chromatographic separations resulted in high yield and purity of adalimumab. The precipitation-based process resulted in high yield but with lower purity. The extraction-based purification resulted in low yield and purity. Thus, the precipitation-based method proved to be more promising than the extraction-based method for direct purification of adalimumab from harvested cell culture fluid. Novelty and scientific contribution. Although alternative purification methods may offer the advantages of simplicity and low-cost operation, further significant improvements are required to compete with the performance of chromatographic separations of adalimumab from true fermentation broth.Pozadina istraživanja. Afinitetna kromatografija pri kojoj se monoklonsko protutijelo veže za protein A je već dobro poznata metoda u farmaceutskoj industriji. Međutim, zbog visokih troškova izdvajanja proteina A pomoću kromatografije te poteškoća u provođenju kontinuiranog procesa važno je ispitati alternativne metode pročišćavanja. Eksperimentalni pristup. U ovome su radu razvijene i optimirane metode ekstrakcije sa ponovnom ekstrakcijom te taloženja i otapanja. Rezultati tih dvaju metoda, odnosno prinos monoklonskih protutijela, količina preostalih nečistoća, kao što su DNA i proteini stanica domaćina, te količina agregata monoklonskih protutijela, uspoređeni su s onima dobivenim afinitetnom kromatografijom na ionskom izmjenjivaču. Za sveobuhvatnu usporedbu različitih metoda, ispitivanja su provedena na identičnim hranjivim podlogama koje su sadržavale adalimumab. Rezultati i zaključci. Afinitetnom kromatografijom na ionskom izmjenjivaču dobiveni su veliki prinos i velika čistoća adalimumaba. Taloženjem su dobiveni veliki prinos, ali manja čistoća uzorka. Pročišćavanje pomoću ekstrakcije rezultiralo je manjim prinosom i manjom čistoćom uzorka. Zaključeno je da je metoda taloženja bolja od ekstrakcije za izravno pročišćavanje adalimumaba iz suspenzije stanične kulture. Novina i znanstveni doprinos. Iako alternativne metode imaju neke prednosti, kao što su jednostavna i jeftina primjena, neophodno ih je unaprijediti da bi dobiveni rezultati bili usporedivi s rezultatima dobivenim pročišćavanjem adalimumaba ionskom kromatografijom u hranjivoj podlozi